The present invention relates to a biosensor for rapid quantification of a substrate contained in a sample with high accuracy.
Conventionally, methods using polarimetry, colorimetry, reductimetry and a variety of chromatography have been developed as the measure for quantitative analysis of sugars such as sucrose and glucose. However, those conventional methods are all poorly specific to sugars and hence have poor accuracy. Among them, the polarimetry is simple in manipulation, but it is largely affected by the temperature during the manipulation. Therefore, this method is not suitable for simple quantification of sugars at home by ordinary people.
In recent years, a variety of biosensors have been developed which best utilize a specific catalytic action of enzymes.
In the following, a method of quantitative analysis of glucose will be explained as an example of the method for quantifying a substrate contained in a sample. Conventionally known electrochemical quantification of glucose includes a method using a combination of glucose oxidase (EC 1.1.3.4: hereinafter abbreviated to “GOD”) as an enzyme with an oxygen electrode or a hydrogen peroxide electrode (see “Biosensor” ed. by Shuichi Suzuki, Kodansha, for example).
GOD selectively oxidizes β-D-glucose as a substrate to D-glucono-δ-lactone using oxygen as an electron mediator. Oxygen is reduced to hydrogen peroxide during the oxidation reaction by GOD in the presence of oxygen. A decreased volume of oxygen is measured by the oxygen electrode, or an increased volume of hydrogen peroxide is measured by the hydrogen peroxide electrode. The decreased volume of oxygen or, otherwise, the increased volume of hydrogen peroxide is proportional to the content of glucose in the sample. It is therefore possible to quantify glucose based on the decreased volume of oxygen or the increased volume of hydrogen peroxide.
In the above method, it is possible to quantify glucose in the sample accurately by using the specificity of the enzyme reaction. However, as speculated from the reaction, this prior art method has a drawback that the measurement result is greatly affected by the oxygen concentration in the sample. Hence, in the event where oxygen is absent in the sample, measurement is infeasible.
Under such a circumstance, a glucose sensor of new type has been developed which uses as the electron mediator an organic compound or a metal complex such as potassium ferricyanide, a ferrocene derivative and a quinone derivative, in place of oxygen in the sample. The sensor of this type oxidizes the reduced electron mediator resulting from the enzyme reaction on a working electrode so as to determine the glucose concentration in the sample based on an oxidation current produced by the oxidation reaction. At this time, on a counter electrode, the oxidized electron mediator is reduced, and a reaction for generating the reduced electron mediator proceeds. With the use of such an organic compound or metal complex as the electron mediator in place of oxygen, it is possible to form a reagent layer by precisely placing a known amount of GOD together with the electron mediator in their stable state on the electrode, thereby enabling accurate quantification of glucose without being affected by the oxygen concentration in the sample. In this case, it is also possible to integrate the reagent layer containing the enzyme and electron mediator with an electrode system while keeping the reagent layer in an almost dry state, and therefore a disposable glucose sensor based on this technology has recently been noted considerably. A typical example of such a glucose sensor is a biosensor disclosed in Japanese Laid-Open Patent Publication Hei 3-202764. With such a disposable glucose sensor, it is possible to measure the glucose concentration easily with a measurement device by simply introducing a sample into the sensor connected detachably to the measurement device. The application of such a technique is not limited to quantification of glucose and may be extended to quantification of any other substrate contained in the sample.
However, in the above-described conventional biosensors, when the sample contains a substrate at high concentrations, the enzyme reaction proceeds also on the counter electrode and supply of the electron mediator to the counter electrode thus becomes insufficient, so that the reaction at the counter electrode becomes a rate determining step, which makes it impossible to obtain a current response proportional to the substrate concentration. Therefore, such biosensors have a problem that quantification of a substrate is not possible when the sample contains a substrate at high concentrations.
In recent years, there is a demand for a biosensor exhibiting a low response when the substrate concentration is zero and excellent storage stability. The response obtained when the substrate concentration is zero is hereinafter referred to as “blank response”.